A survey of the distribution of the PKD-parasite Tetracapsuloides bryosalmonae (Cnidaria: Myxozoa: Malacosporea) in salmonids in Norwegian rivers - additional information gleaned from formerly collected fish.

The malacosporean Tetracapsuloides bryosalmonae was detected in kidneys from Atlantic salmon parr in 64 of 91 sampled Norwegian rivers. Using real-time PCR, this parasite was found to be present in Atlantic salmon parr in rivers along the whole coast, from the northernmost and southernmost areas of the country. In addition, T. bryosalmonae was found in kidneys from brown trout parr in 17 of 19 sampled rivers in south-east Norway, and in Arctic charr sampled in the River Risfjordelva, located at the northernmost edge of the European mainland. In conclusion, T. bryosalmonae has a widespread distribution in salmonids in Norwegian watercourses. Proliferative kidney disease (PKD) caused by T. bryosalmonae and PKD-induced mortality has been observed in salmonids in several Norwegian rivers and it can be speculated that more PKD outbreaks will occur as a result of climate change.

Morphological diversity of Paramoeba perurans trophozoites and their interaction with Atlantic salmon, Salmo salar L., gills.

Amoebic gill disease (AGD) caused by the ectoparasite Paramoeba perurans affects several cultured marine fish species worldwide. In this study, the morphology and ultrastructure of P. perurans in vitro and in vivo was investigated using scanning and transmission electron microscopy (SEM and TEM, respectively). Amoebae cultures contained several different morphologies ranging from a distinct rounded cell structure and polymorphic cells with pseudopodia of different lengths and shapes. SEM studies of the gills of AGD-affected Atlantic salmon, Salmo salar L., revealed the presence of enlarged swellings in affected gill filaments and fusion of adjacent lamellae. Spherical amoebae appeared to embed within the epithelium, and subsequently leave hemispherical indentations with visible fenestrations in the basolateral surface following their departure. These fenestrated structures corresponded to the presence of pseudopodia which could be seen by TEM to penetrate into the epithelium. The membrane-membrane interface contained an amorphous and slightly fibrous matrix. This suggests the existence of cellular glycocalyces and a role for extracellular products in mediating pathological changes in amoebic gill disease.

Presence of Anisakis simplex (Rudolphi, 1809 det. Krabbe, 1878) and Hysterothylacium aduncum (Rudolphi, 1802) (Nematoda; Anisakidae) in runts of farmed Atlantic salmon, Salmo salar L.

One hundred farmed Atlantic salmon, Salmo salar L., were examined for the presence of nematodes by digestion of tissue in HCl-pepsin solution. All fish were sampled from one cage in a fish farm on the Norwegian south-west coast. Fifty harvest quality salmon, that is, salmon for human consumption (mean 5.4 kg, variation 3.0-7.6 kg), were sampled at the processing line while 50 salmon runts (mean 1.1 kg, variation 0.4-1.8 kg), discarded due to poor performance, were sampled from the discard bin after the grading station. Runts are individual fish with clear signs of poor performance over time and abnormal appearance and are thus not processed for human consumption. No nematodes were found in the musculature or viscera of the 50 harvest quality salmon. In total, 75 nematodes were found in 10 (20%) of the runts; 53 nematodes in the viscera and 22 in the musculature. Nematodes in the musculature were identified as Anisakis simplex (Rudolphi, 1809 det. Krabbe, 1878), while nematodes in the viscera were identified as A. simplex and Hysterothylacium aduncum (Rudolphi, 1802).

Early marine migration of European silver eel Anguilla anguilla in northern Norway.

To study the migratory behaviour in wild northern European silver eel Anguilla anguilla during sea entry and early marine migration, 32 individuals were tagged with acoustic transmitters and registered at four automatic listening station arrays from the mouth of the north Norwegian River Alta and throughout the Alta Fjord. The A. anguilla entered the fjord during all parts of the tidal cycle and did not seem to utilize the outgoing tidal currents. They migrated mainly during the night, in both the river mouth and the fjord. On average, they spent 2·7 days travelling from the river mouth to the outermost array, 31 km from the river mouth, corresponding to an average migratory speed of 0·5 km h(-1) . The A. anguilla generally migrated in the central part of the fjord and in the uppermost 10-25% of the water column, but with frequent dives to greater depths. Already 4 km after sea entry, A. anguilla were observed diving deeper than 130 m within 20-30 min periods. Hence, this study demonstrated that A. anguilla may perform an active diving behaviour during the early marine migration. The study took place in a pristine area with a minimum of anthropogenic interventions and by individuals from a population still uninfected by the introduced parasite Anguillicoloides crassus. The results may therefore be used as a baseline for future studies of the A. anguilla early marine migration.

Multi-centre testing and validation of current protocols for the identification of Gyrodactylus salaris (Monogenea).

Despite routine screening requirements for the notifiable fish pathogen Gyrodactylus salaris, no standard operating procedure exists for its rapid identification and discrimination from other species of Gyrodactylus. This study assessed screening and identification efficiencies under real-world conditions for the most commonly employed identification methodologies: visual, morphometric and molecular analyses. Obtained data were used to design a best-practice processing and decision-making protocol allowing rapid specimen throughput and maximal classification accuracy. True specimen identities were established using a consensus from all three identification methods, coupled with the use of host and location information. The most experienced salmonid gyrodactylid expert correctly identified 95.1% of G. salaris specimens. Statistical methods of classification identified 66.7% of the G. salaris, demonstrating the need for much wider training. Molecular techniques (internal transcribed spacer region-restriction fragment length polymorphism (ITS-RFLP)/cytochrome c oxidase I (COI) sequencing) conducted in the diagnostic laboratory most experienced in the analysis of gyrodactylid material, identified 100% of the true G. salaris specimens. Taking into account causes of potential specimen loss, the probabilities of a specimen being accurately identified were 95%, 87% and 92% for visual, morphometric and molecular techniques, respectively, and the probabilities of correctly identifying a specimen of G. salaris by each method were 81%, 58% and 92%. Inter-analyst agreement for 189 gyrodactylids assessed by all three methods using Fleiss' Kappa suggested substantial agreement in identification between the methods. During routine surveillance periods when low numbers of specimens are analysed, we recommend that specimens be analysed using the ITS-RFLP approach followed by sequencing of specimens with a "G. salaris-like" (i.e. G. salaris, Gyrodactylus thymalli) banding pattern. During periods of suspected outbreaks, where a high volume of specimens is expected, we recommended that specimens be identified using visual identification, as the fastest processing method, to select "G. salaris-like" specimens, which are subsequently identified by molecular-based techniques.

Occurrence of postmortem myoliquefactive kudoosis in Atlantic mackerel, Scomber scombrus L., from the North Sea.

Members of the myxosporean genus Kudoa occur in various marine teleosts worldwide. Several species are of concern to the fishery and aquaculture industries as they may produce unsightly cysts in the fish host's musculature or are associated with postmortem myoliquefaction of the fish muscle, commonly referred to as 'soft flesh'. This study describes the occurrence and effects on a host of a Kudoa species in Atlantic mackerel, Scomber scombrus, from the northern North Sea. Generalized postmortem myoliquefaction associated with Kudoa sp. occurred in 0.8% of the examined fish, i.e. 11 of 1339 mackerel developed 'soft flesh'. There was a significant difference in the prevalence of myoliquefaction between medium sized (400-600 g) and large mackerel (>600 g). The prevalence reached 8.9% in the latter host size group. No subclinical infections of Kudoa sp. were detected when examining fresh muscle (n = 103) and blood (n = 165) samples for spores using light microscopy. Affected mackerel developed generalized myoliquefaction after 38-56 h post-catch. No inflammatory host response was associated with the presence of plasmodia within single body muscle fibres of 'soft flesh' affected fish. Based on comparison of myxospore dimensions and analysis of the nuclear small subunit (SSU) ribosomal DNA, the present Kudoa species is assigned to Kudoa thyrsites. However, due to the species' apparently very wide geographical distribution and host range, its varying effect on different fish host species, together with the still unknown life cycle of Kudoa spp., the taxonomic status of K. thyrsites appears not to be fully resolved.

The mitochondrial genome of Gyrodactylus salaris (Platyhelminthes: Monogenea), a pathogen of Atlantic salmon (Salmo salar).

In the present study, we describe the complete mitochondrial (mt) genome of the Atlantic salmon parasite Gyrodactylus salaris, the first for any monogenean species. The circular genome is 14,790 bp in size. All of the 35 genes recognized from other flatworm mitochondrial genomes were identified, and they are transcribed from the same strand. The protein-coding and ribosomal RNA (rRNA) genes share the same gene arrangement as those published previously for neodermatan mt genomes (representing cestodes and digeneans only), and the genome has an overall A+T content of 65%. Three transfer RNA (tRNA) genes overlap with other genes, whereas the secondary structure of 3 tRNA genes lack the DHU arm and 1 tRNA gene lacks the TphiC arm. Eighteen regions of non-coding DNA ranging from 4 to 112 bp in length, totalling 278 bp, were identified as well as 2 large non-coding regions (799 bp and 768 bp) that were almost identical to each other. The completion of the mt genome offers the opportunity of defining new molecular markers for studying evolutionary relationships within and among gyrodactylid species.

The effect of various metals on Gyrodactylus salaris (Platyhelminthes, Monogenea) infections in Atlantic salmon (Salmo salar).

Atlantic salmon (Salmo salar) parr (age 0+), infected by the ectoparasite Gyrodactylus salaris, were exposed to aqueous aluminium (Al), copper (Cu), zinc (Zn), iron (Fe) and manganese (Mn), at 4 different concentrations. There was a negative correlation between G. salaris infections and metal concentrations in both Zn- and Al-exposed salmon. In the Zn-experiment, all 4 concentrations tested caused a decrease in the G. salaris infections, while in the Al-experiment the G. salaris infection did not decline at the lowest concentration. The number of G. salaris increased continuously during the experiments in all control groups, and in all groups exposed to Cu, Fe and Mn. At the highest concentration, however, copper seemed to impair the growth of G. salaris infection. The results show that aqueous Al and Zn are environmental factors of importance controlling the distribution and abundance of the pathogen G. salaris. Other pollutants might also have an influence on the occurrence of G. salaris. Finally, the results demonstrate that aqueous Al and Zn have a stronger effect on the parasite than on the salmonid host, suggesting that both metals may be used as a pesticide to control ectoparasites such as G. salaris.

Analysis of ribosomal RNA intergenic spacer (IGS) sequences in species and populations of Gyrodactylus (Platyhelminthes: Monogenea) from salmonid fish in Northern Europe.

The intergenic spacer (IGS) region of ribosomal RNA genes was amplified and sequenced from a variety of Gyrodactylus specimens collected from wild and farmed Atlantic salmon Salmo salar, rainbow trout Oncorhynchus mykiss, and grayling Thymallus thymallus, from various locations in Northern Europe. Phylogenetic analysis of the sequences confirmed the distinction between G. salaris Malmberg, 1957 and G. thymalli Zitnan, 1960, supporting their validity as separate species. G. salaris adapted to rainbow trout are also distinct from the parasites found on Atlantic salmon, supporting the existence of a rainbow-trout form that was initially identified on the basis of morphological differences. Analysis of the IGS did not provide good resolution of different populations of G. salaris sensu stricto, but was consistent with epidemiological evidence which indicates that introduction of the parasite to Norway was recent and limited. The IGS may be helpful in distinguishing forms of G. salaris that are pathogenic to Atlantic salmon from those that are not.

The use of host specificity, pathogenicity, and molecular markers to differentiate between Gyrodactylus salaris Malmberg, 1957 and G. thymalli Zitnan, 1960 (Monogenea: Gyrodactylidae).

The validity of Gyrodactylus thymalli has been questioned, based on its morphological and genetic resemblance to G. salaris. This taxonomic problem has practical implications regarding correct diagnosis of G. salaris, which has proved to be highly pathogenic to stocks of wild Norwegian Atlantic salmon. The host specificity and pathogenicity of G. salaris and G. thymalli were experimentally tested on salmon and grayling. Both parasite species were able to infect, live and reproduce on both fish species. G. salaris was highly pathogenic for the experimental stock of salmon, while grayling mounted an effective response against this parasite. Both fish species responded to an infection with G. thymalli. The results did not support the hypothesis of conspecificity between G. thymalli and G. salaris. The ribosomal RNA gene intergenic spacer of both species was sequenced. Variation in sequence was lower than expected for different species. Variation in the sequences of tandemly repeated elements was found and may prove useful in distinguishing G. salaris and G. thymalli.

A model of salmon louse production in Norway: effects of increasing salmon production and public management measures.

Salmon lice Lepeophtheirus salmonis Krøyer have caused disease problems in farmed Atlantic salmon Salmo salar L. since the mid-1970s in Norway. High infection intensities and premature return of wild sea trout Salmo trutta L. were first reported in 1992. Later emaciated wild Atlantic salmon smolts carrying large amounts of lice have been observed both in fjords and offshore. The Norwegian Animal Health Authority regulations to control the problem, which came into operation in 1998, included compulsory louse level monitoring in farms and maximum legal numbers of lice per fish. Here, we present a model of salmon louse egg production in Norway and show that the effect of the current public management strategy is critically dependent on the yearly increase in salmon production. This is because the infection pressure is the product of the number of fish in the system, and the number of lice per fish. Due to the much larger number of farmed than wild salmonids, it is highly likely that lice originating from farmed salmon infect wild stock. Estimated tolerance limits for wild salmonids vary widely, and the level of louse egg production in farms which would be needed to decimate wild populations is not known. Two possible thresholds for total lice egg production are investigated: (1) 1986 to 1987 level (i.e. before adverse effects on sea trout were recorded), and (2) a level corresponding to a doubling of the estimated natural infection pressure. The farm lice per fish limits that would have to be observed to keep louse production within the 2 thresholds are calculated for the period 1986 to 2005. A steady decrease in the permitted number of lice per fish may keep the total louse production stable, but the number of salmon required for verification of lice numbers will increase as the prevalence to be verified is decreased. At threshold (2), the model estimated that lice limits should have been 0.05 louse per fish in 1999. This would require 60 fish from each pen to be collected, anaesthetised and examined for a good estimate at a confidence level of 95%. Such sample numbers are likely to be opposed by farmers. The use of national delousing programs to solve the problem is discussed.

Redescription of Gyrodactylus teuchis Lautraite, Blanc, Thiery, Daniel & Vigneulle, 1999 (Monogenea: Gyrodactylidae); a species identified by ribosomal RNA sequence.

Molecular and morphological features of Gyrodactylus specimens from Oncorhynchus mykiss, Salmo trutta and Salmo salar were examined. Sequences from variable region V4 of the small subunit ribosomal RNA gene and the ribosomal RNA internal transcribed spacers, produced by the FRS Marine Laboratory, revealed that these were not the same as other well-characterised Gyrodactylus that are common on European salmonids and were in fact a distinct species. Initial morphological examination of the opisthaptor indicated that this species very closely resembles G. salaris Malmberg, 1957. More detailed analysis revealed differences in the shape of the marginal hook sickle of these two species and thus Gyrodactylus teuchis Lautraite, Blanc, Thiery, Daniel & Vigneulle, 1999 was erected. Analysis of the ribosomal RNA gene or spacer sequences remains the most reliable method of identifying this species. This is believed to be the first record of a Gyrodactylus species identified first from molecular data and confirmed by morphological examination; previous molecular analyses had provided alternative methods for identifying species that had already been described using morphological characters.

A Monte Carlo simulation model for assessing the risk of introduction of Gyrodactylus salaris to the Tana river, Norway.

The Tana river in northern Norway, the most productive salmon river in Europe, is free of Gyrodactylus salaris. Currently there is one salmon farm in operation on the Tana fjord. Because of the strong association between stocking of rivers with salmon and infestations with G. salaris there is national and international concern that the existing farm might lead to the introduction of the parasite to the Tana river. In response to these concerns a quantitative analysis of the risk of introduction of G. salaris to the Tana river was undertaken. A scenario tree, the Monte Carlo simulation model and results of the simulations including sensitivity analyses are presented and discussed. Results show that the probability of introduction of G. salaris to the Tana river via transfer of smolt to the existing salmon farm is extremely low primarily due to the low probability that the transferred smolt become infested. The total risk was very sensitive to changes in the salinity of the water at the sea site.

Systemic spironucleosis in sea-farmed Atlantic salmon Salmo salar, caused by Spironucleus barkhanus transmitted from feral Arctic char Salvelinus alpinus?

A hexamitid flagellate was found in the gall bladder and intestine of Arctic char Salvelinus alpinus in northern Norway. Scanning and transmission electron microscopy showed this flagellate to be identical to Spironucleus barkhanus from grayling Thymallus thymallus and farmed Atlantic salmon Salmo salar. It is hypothesized that systemic spironucleosis in sea-caged Atlantic salmon was due to transmission of flagellates from feral char to the salmon.

Seasonal occurrence of Gyrodactylus derjavini (Monogenea) on brown trout, Salmo trutta, and Atlantic salmon, S. salar, in the Sandvikselva river, Norway.

Seasonal variation in prevalence and intensity of the ectoparasite Gyrodactylus derjavini on brown trout parr, Salmo trutta, and Atlantic salmon parr, S. salar, and occurrence of G. derjavini on ascending sea trout, S. trutta m. trutta, in the Sandvikselva river, southeastern Norway, was recorded. In general, both prevalence and intensity increased and decreased correspondingly with the rise and fall in water temperature. However, both prevalence and intensity decreased in warm periods when reproduction and transmission should be high, possibly from host-induced parasite mortality. Sea trout became infected with G. derjavini soon after they entered the river; prevalence tended to increase as the sea trout migrated upstream to the spawning grounds. It is hypothesized that transmission of G. derjavini to sea trout occurred via the river substratum.

The effect of Gyrodactylus salaris (Monogenea) on the epidermis of Atlantic salmon, Salmo salar, parr in the river Batnfjordselva, Norway.

Skin morphology of Atlantic salmon, Salmo salar, parr infected with Gyrodactylus salaris was examined for 3 yr in the river Batnfjordselva in Norway and compared to that of uninfected salmon parr from a neighboring river. The epidermis of the infected population had more cell layers and was thicker than the epidermis of parr from the uninfected population. The number of mucous cells did not differ, and no seasonal changes in morphology of the epidermis were detected in either rivers. Intensity of G. salaris did not correlate to epidermal thickness, epidermal cell layers, or mucous cell concentration.

Random amplified polymorphic DNA (RAPD) analysis of three Norwegian Gyrodactylus salaris populations (Monogenea:Gyrodactylidae).

Gyrodactylus salaris Malmberg, 1957 from Atlantic salmon (Salmo salar L.) parr in 3 Norwegian rivers have been compared genetically by the use of random amplified polymorphic DNA (RAPD) analysis. Two primers revealed polymorphism between different populations of G. salaris. This variation has probably arisen since the introduction of G. salaris to Norway in the 1970s and complements results showing morphological variation between G. salaris from different geographical areas.

Population dynamics of Gyrodactylus salaris (Monogenea) infecting Atlantic salmon, Salmo salar, parr in the river Batnfjordselva, Norway.

Population dynamics of Gyrodactylus salaris on 5 year classes of Atlantic salmon, Salmo salar, in the river Batnfjordselva in Norway was studied from April 1991 to June 1994. Prevalence was 100% on all year classes throughout the study period, except for a marked decline in winter and spring of 1992. Median abundance generally increased during summer and peaked in September/October at levels between 400 and 1,300 parasites. The lowest number of parasites was observed during winter and spring. The seasonal dynamics in the first year of the study differed markedly from the remainder of the study, abundance peaked already in June/July on 1+ and 2+ fish the first year, but decreased throughout summer and peaked again in autumn. A collapse in the G. salaris population was observed in the winter of 1991/1992, with prevalence being 0-40% on all year classes. Parr aged 2 yr harbored significantly fewer parasites than younger parr during the winter months. Parasite intensity on precocious males was significantly higher than on sexually immature males and females. Gyrodactylus salaris was found on the fins (76%), gill filaments (15%), and head/body (9%). During the last 2 yr of the study the gills were increasingly infected, with 40% of the parasites found on gills in some samples.